Improved structural preservation and immunolocalization of the microtubule cytoskeleton in plant and animal cells by freeze substitution/fixation

Abstract

Immunofluorescence localization is widely used to determine the distribution and organization of the microtubule (Mt) cytoskeleton in cells, with most protocols using conventional chemical fixation (CCF). However, concerns associated with CCF are slow rate of fixation, alteration of cell morphology, and redistribution of target antigens. Freeze substitution/fixation (FSF) is a method commonly used in electron microscopy to overcome these problems, but has not been exploited at the light microscopic level. Thus, techniques using FSF were investigated in three divergent cell types (tobacco suspension cultures, Tradescantia pollen tubes, and mouse myeloma cultures) and evaluations were made of cell morphology and immunofluorescence localization of the Mt cytoskeleton. Tobacco cells prepared by FSF exhibited an extensive network of Mts with consistent and strong labeling, while CCF preparations had disorganized and fuzzy Mts. Pollen prepared with FSF retained cytoplasmic details, and had continuous longitudinal arrays of cortical Mts. In contrast, CCF induced structural abnormalities in pollen characterized by swollen and ruptured tube tips with sinuous, nonuniformly labeled Mts. Likewise, in myeloma cells, Mt labeling was more uniform and stronger with less background with FSF versus CCF. CCF also caused myelomas to shrink. Advantages of FSF over CCF are that rapid freezing attains an instantaneous cessation of metabolism with better preservation of cellular structures, and enhanced and more consistent Mt labeling. The absence of numerous amendments used in CCF (e.g., buffers and osmotic reagents) may avoid interaction with target antigens. FSF preparations should be useful in critical immunofluorescence localizations of other antigens, especially those sensitive to CCF manipulations.